Method for sensing the concentration of glucose in biological fluids

ABSTRACT

Glucose sensing apparatus for use in biological fluids comprising an electrode and a voltage source connected to the electrode to cause a current to flow therethrough, in which the voltage applied to the electrode is varied in a sweep ranging from a first limit to a second limit and back toward the first limit, and the current at the electrode is integrated with respect to time during at least a portion of the voltage variation selected to produce a total charge sensitive to glucose concentration and relatively insensitive to other components in the fluid; alternatively the current is measured at a point in a specific region of the voltage variation.

This is a division of application Ser. No. 155,198, filed June 2, 1980 and now U.S. Pat. No. 4,340,458.

BACKGROUND OF THE INVENTION

This invention relates to sensing the concentration of glucose in biological fluids.

It is desirable to monitor accurately glucose concentration in biological fluids, particularly using a device that is implantable in living organisms; for example, such a device would be useful for diabetics in timing the administration of insulin and food.

Glucose concentration in simple solutions--e.g., Krebs-Ringer buffer--can be monitored electrochemically by measuring the current density at a platinum electrode while varying the electrode potential relative to a standard. At various electrode potentials, current generated by electrochemical reactions is responsive to the glucose concentration. Accordingly, current measured while varying the electrode potential over a range, first from negative to positive (anodic) and then back again (cathodic), will be a function of glucose concentration.*

Interference with glucose measurement due to the presence of other biological substances--particularly amino acids and urea--is a serious problem. Such substances undergo electrochemical reactions during the voltage sweep, generating currents independent of glucose concentration and reducing sensitivity to glucose.

Marincic et al., infra, indicates that there is considerable sensitivity of current density to glucose concentration in the presence of urea with cathodic potential sweeps at potentials below about -100 mV; that article concludes, however, for anodic potential sweeps, that inhibition by urea precludes glucose concentration measurement.

Gebhart et al., "Development of an Implantable Electrocatalytic Glucose Sensor", 5 Bioelectrochemistry and Bioenergetics 607 (1978) indicates (FIG. 9) a correlation between glucose concentration in the presence of amino acids and charge produced at the electrode at a specific voltage; between periods of integration, voltage is shifted to a rejuvenation potential.

SUMMARY OF THE INVENTION

According to one aspect of the invention, by applying a voltage to the electrode, varying the voltage in a sweep from a first limit to a second limit and back toward the first limit, and integrating the current at the electrode over at least a portion of the voltage sweep selected to produce a total charge sensitive to glucose concentration and to be relatively insensitive to other components in the biological fluid, an accurate measure of glucose concentration is obtained in biological fluids.

In preferred embodiments, the portion of integration is selected so that the charge responsive to glucose concentation has predominantly the same algebraic sign throughout the portion, and so that the charge responsive to interfering components of the fluid (or to any specific interfering component) has a positive sign in one segment of the portion and a negative sign in another segment, so that the glucose-responsive charge, and thus sensitivity to glucose, is reinforced throughout the portion of integration, and the charge responsive to interfering substances at least partially cancels, thus reducing interference from those components or changes in concentration of those components. Total charge is normalized in the form of the ratio of total algebraic charge to total absolute value of charge; the lower limit of the voltage sweep is between -1.0 and -0.6 volts and the upper limit is between 0.7 and 1.1 volts; the voltage sweep includes dwell periods at the limits, and the rates of voltage change during the anodic and cathodic sweeps are constant; integration takes place continuously during one complete voltage sweep (or, alternatively, substantially including the anodic sweep between -0.45 volts and the upper limit, the dwell at the upper limit, and the entire cathodic sweep); the rate of voltage change during cathodic voltage sweep may be more rapid than during the anodic voltage sweep; and the voltage sweep may include holding periods between the limits at potentials at which glucose undergoes an electrochemical reaction.

According to another aspect of the invention, glucose concentration in biological fluids can be determined by measuring the current at the electrode at a point in a specific region between 0.2 volts and 0.6 volts during the anodic voltage variation selected to be sensitive to changes in levels of glucose concentration but relatively insensitive to levels of other constituents of the fluid.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

We turn now to a description of the preferred embodiments of the invention, first briefly describing the drawings thereof.

Drawings

FIG. 1 is a block diagram of the circuitry for an implantable glucose sensor embodying one aspect of the invention.

FIG. 2 is a block diagram of the circuitry for an implantable glucose sensor embodying another aspect of the invention.

FIGS. 3a and 3b show, diagramatically, the elements of an electrode cell used with the embodiments of FIG. 1 or 2.

FIGS. 4a, b, c, d, e and f are graphs related to the embodiment of FIG. 1.

FIGS. 5a, b, and c are graphs related to the embodiment of FIG. 2.

INTEGRATION OF CURRENT

FIG. 1 shows in block form, one embodiment of implantable circuitry including glucose sensing cell 40. FIGS. 3a and 3b show potentiostat 30 connected to control the potential drop between glucose oxidation electrode 50 and reference elecrode 52. Specifically, potentiostat 30 compares and equates that voltage (V meas.) to the voltage (V appl.) generated by function generator 20 by allowing current to flow in a measurement circuit formed by leads 51, glucose electrode 50, the biological medium, counter electrode 54, lead 53 and potentiostat 30. Reference electrode 52 is connected to the function generator by lead 55.

In FIG. 1, signal processor 60 is connected to the measurement circuit and integrates current in the circuit in response to timed signals from function generator 20. Modulator/transmitter 70 is connected to signal processor 60 and transmits an FM signal corresponding to the output of processor 60 in response to a transmit signal from function generator 20. Sensor cell 40 (FIG. 3) has a glucose oxidation electrode 50 separated by barrier 56 (which is an ion exchange membrane treated to allow passage of cations only) from counter electrode 54. A glucose-permeable membrane 58 separates electrode 50 from high molecular weight compounds in the biological medium, and a biocompatible hydrophobic membrane 59 at the counter electrode allows reaction products (gasses) to diffuse to surrounding tissue, while electrically insulating the electrode from that tissue. Reference electrode 52 is a silver/silver chloride electrode. Counter electrode 54 is a carbon electrode.

Glucose electrode 50 is a teflon-bonded platinized platinum electrode prepared as follows: An electrode sheet is formed from a suspension of Teflon-30 (Dupont) and platinum black (Engelhard), respectively. This suspension is then filtered through a membrane to give a uniform layer of the Teflon and platinum on the membrane. This layered structure is then placed atop a platinum mesh screen with the Teflon-platinum layer mixture contacting the platinum screen. The resultant layered structure is subjected to pressure and then sintered to obtain the final electrode structure. The electrode is cut from the sheet.

In operation, function generator 20 generates a waveform which varies back and forth between two limits. Line 2 in FIG. 4a shows a typical trapezoidal waveform in which voltage is maintained at -0.8 V for 60 sec., then increased at a rate of 2 volts/min. to +0.8 V. This region of increasing voltage is called the anodic voltage sweep. After a 30 second dwell at 0.8 V, voltage is decreased, again at 2 volts/min. to the lower limit of -0.8 V. The region of decreasing voltage is the cathodic voltage sweep. By selecting a voltage sweep which effectively permits glucose oxidation and rejuvenates the catalytic surface of the electrode, continuous sweeps and glucose concentration measurement are possible.

As the voltage is applied to the electrode, electrochemical reaction of glucose is catalyzed at electrode 50. Glucose from the surrounding tissue permeates the membrane, and reaction products are transported away from the electrode. Barrier 56 insulates the two electrodes, but allows transfer of cations to the counter electrode to complete the current measurement circuit.

The biological medium produces currents at specific portions of the voltage sweep. In particular glucose oxidation occurs at specific portions of the sweep; in those portions, the current generated (which will include current generated as a result of reaction of background constituents as well as glucose-responsive current) can be used to indicate glucose concentration. However, electrochemical reactions due to other fluid constituents--specifically urea and amino acids--may affect the current measurement and hinder determination of glucose concentration.

In certain portions of the sweep, the current related to a particular interfering substance is positive in one segment of the portion and negative in another segment, while the effect of glucose-related currents is essentially positive throughout the portion. By integrating cell current over time to obtain total charge at the electrode in that sweep portion, these glucose-reinforcing, interference-cancelling phenomena act to improve greatly the sensitivity to glucose concentration while at the same time reducing the interference experienced. Specifically, signal processor 60 integrates current and provides a measurement of total algebraic charge Q_(t) over the region of integration; alternatively the signal processor can provide a signal representing ratio of the total algebraic charge to the absolute value of total charge Q_(t) /|Q_(t) |.

The graphs of FIG. 4 represent examples of operations conducted under simulated physiologic conditions. They are performed at 37° C. under an atmosphere of 95% nitrogen, 5% CO₂. The background fluid is a modified Kreb-Ringer buffer solution (0.109 M NaCl, 0.024 M NaHCO₃, 0.006 M MgSo₄, 0.001 M NaH₂ PO₄, pH=7.4). Amino acids are provided in a stock solution of 23 different amino acids at concentrations proportional to physiological concentrations. Urea is present in the physiological concentrations indicated. The potentiostat used (Princeton Applied Research Corp., Model PAR-173) has automatic IR compensation. The programmable function generator is Model 73211 from Data Trak, Research, Inc. The glucose electrode has a geometric area of 0.19 cm², a Pt coat of 25 mg per cm² and a real area (from coulometric hydrogen stripping measurement) of 485 cm². The reference electrode is a standard saturated colomel (Hg, Hg₂ Cl₂, KCl) electrode, and the glucose permeable membrane is Nucleopore, 500 Angstrom.

When the above-described voltage waveform (line 2 in FIG. 4a) is applied to the Krebs-Ringer buffer alone, the current at the electrode is first negative, then positive and finally negative, as indicated by line 1 in FIG. 4(a). The shaded area under line 1 represents charge, and the positive and negative areas essentially cancel.

FIG. 4b shows current measured in the system before (line 1) and after (line 2) the addition of glucose. The difference attributable to glucose (line 3) results in a charge (shaded) which is essentially positive over the entire waveform.

With the addition of urea (FIG. 4c) or amino acids (FIG. 4d) to the system, the resulting current changes (line 2 in FIGS. 4c and d) are positive in some regions of the sweep and negative in other regions, while the current responsive to glucose (line 1) remains essentially positive throughout the sweep.

The following table shows that integrating current over the entire sweep provides an algebraic value of charge (Qt) (or a normalized value of charge in the form of the ratio of algebraic to total absolute charge (Q_(t) /Q_(t))) which is sensitive to glucose concentration even in the presence of changing concentrations of amino acids and urea. FIGS. 4e and f specifically illustrate the above relationship in the presence of changing concentrations of amino acids.

    ______________________________________                                         Glucose Electrode Response to Changes in                                       Glucose and Amino Acid Concentration                                           Conc., mg/dl                                                                    Glucose   AcidsAmino                                                                             Urea      mCoulQ.sub.t,                                                                        ##STR1##                                    ______________________________________                                          50       35      40        20.0  .087                                          63       35      40        22.6  .099                                          75       35      40        24.5  .107                                          88       35      40        26.0  .114                                         100       35      40        27.6  .122                                         113       35      40        28.0  .123                                         125       35      40        30.0  .132                                         150       35      40        31.9  .141                                         175       35      40        33.7  .148                                         200       35      40        36.3  .160                                         250       35      40        41.0  .180                                          50       50      40        23.8  .109                                          63       50      40        24.3  .111                                          75       50      40        24.9  .114                                          88       50      40        27.3  .126                                         100       50      40        28.8  .133                                         113       50      40        28.5  .132                                         125       50      40        28.9  .134                                         150       50      40        31.2  .144                                         175       50      40        34.4  .159                                         200       50      40        35.2  .162                                         250       50      40        38.5  .178                                          50       65      40        23.6  .117                                          63       65      40        23.7  .119                                          75       65      40        24.4  .123                                          88       65      40        25.2  .128                                         100       65      40        26.5  .135                                         113       65      40        26.7  .137                                         125       65      40        27.3  .140                                         150       65      40        29.1  .149                                         175       65      40        31.3  .160                                         200       65      40        31.9  .165                                         250       65      40        34.1  .177                                         ______________________________________                                    

Thus, when integrating over the entire voltage sweep, the background charge (from the background solution and from the amino acids and urea) essentially cancels, while the glucose-responsive charge is essentially positive and, sensitivity to glucose is increased by integration. Moreover, changes in charges associated with changes in urea and amino acid concentrations, respectively, essentially cancel, thus enhancing sensitivity to glucose.

Referring specifically to FIGS. 4c and d, it is apparent that integration need not occur over the entire voltage sweep. Examination of a specific portion of the sweep--substantially including the portion of the anodic sweep from -0.45 volts to 0.8 volts, the dwell at the upper limit, and the entire cathodic sweep--also shows the requisite characteristics for selectivity and sensitivity.

CURRENT MEASUREMENT

FIG. 2 shows a diagram of apparatus similar to FIG. 1, except that function generator 20 signals current measurement by current measurement means 80, and transmission of a signal representing that measurement by transmittter 70. FIGS. 5a, b and c represent the results of current measurement, under conditions described for the current integration examples above. The region between 0.2 and 0.6 volts of the anodic portion of the sweep shows a clear response to changes in glucose concentration, and a lack of response to changes in concentrations of amino acids and urea. Measurement at a point within that region (e.g., 0.3 volts) thus provides a measurement of glucose concentrations notwithstanding the presence of urea and amino acids. For example values of current at 0.3 V in the anodic scan for a Krebs-Ringer buffer containing 40 mg/dl of urea and 35 mg/dl of amino acids, taken from a plot such as FIG. 5c, will show a linear relationship between current and glucose concentration.

The embodiments of both FIG. 1 and of FIG. 2 would include a receiver (not shown) outside of the organism in which the sensor is implanted, to receive the signal transmitted by the sensor and translate that signal into data representing glucose concentration. Alternatively the receiver could trigger an alarm when glucose concentration varies outside predetermined limits.

OTHER EMBODIMENTS

Other embodiments are within the scope of the following claims. By way of example only, the voltage waveform may be varied in several ways: the dwell times can be changed but should preferably be at least 2 seconds; the lower limit can be increased to, e.g., -1.0 volts, but excessive generation of hydrogen results from a limit significantly below that level. The upper limit can vary, e.g., from 0.7 to 1.1 volts; a voltage significantly above 1.1 V causes corrosion of the electrode. Holding periods may be introduced during the anodic and/or the cathodic portion of the sweep, preferably at potentials at which glucose reactions occur so as to increase the amount of time spent at such potentials and enhance sensitivity and selectivity to glucose; the rate of voltage change in the anodic and cathodic sweeps may be different--e.g., the anodic sweep may be relative slower to increase the amount of time spent at voltages which produce currents sensitive and selective for glucose. With respect to the current measurement embodiment of FIG. 2, the voltage variation could include discontinuities such as jumps from a limit to the measuring voltage.

The claims include glucose sensors that are not implanted--e.g., clinical laboratory sensors. The biological fluids can include any fluids taken from living organisms or fluids simulating conditions in a living organism including, without limitation, blood, urine, buffer solutions, and saline solutions. 

We claim:
 1. A method for sensing the concentration of glucose in biological fluids comprising the steps ofapplying a variable voltage to an electrode to cause a current flow therethrough sweeping said voltage over a range in a first direction from a first limit to a second limit and in the reverse direction back toward said first limit, and integrating current at the electrode with respect to time during a period including at least a portion of said sweep in both directions selected to produce a total charge sensitive to glucose concentration and relatively insensitive to other components in the fluid.
 2. The method of claim 1 wherein said portion is selected so that the charge responsive to glucose concentration has predominantly the same algebraic sign throughout said period, and so that the charge responsive to interfering components of said fluid has a positive sign in one segment of said period and a negative sign in another segment of said period, whereby the glucose-responsive charge, and thus sensitivity to glucose, is reinforced, and the charge responsive to interfering substances at least partially cancels, thus reducing such interference.
 3. The method of claim 1 or 2 wherein the lower limit of such voltage sweep is between -1.0 and -0.6 volts and the upper limit is between 0.7 and 1.1 volts, said sweep including an anodic sweep from said lower to said upper limit and a cathodic sweep from said upper to said lower limit.
 4. The method of claim 1 or 2 wherein said voltage sweep includes dwell periods at said upper and lower limits, and the rate of voltage change during said anodic and cathodic sweeps is constant.
 5. The method of claim 1 or 2 wherein said integration takes place continuously during one complete voltage sweep.
 6. The method of claim 1 or 2 wherein said integration occurs substantially including the anodic sweep between -0.45 volts and said upper limit, the dwell at said upper limit, and the entire cathodic voltage sweep.
 7. The method of claim 1 or 2 wherein the rate of voltage change during said cathodic voltage sweep is more rapid than during said anodic voltage sweep.
 8. The method of claim 1 or 2 wherein said varying of said applied voltage ceases during holding periods between said limits, said holding periods being at potentials at which glucose undergoes an electrochemical reaction.
 9. The method of claim 1 or 2 wherein said portion is selected so that the charge attributable to any specific interfering component of the fluid cancels, whereby changes in concentration of that specific component do not affect the charge measured.
 10. The method of claim 9 wherein said voltage variation includes an anodic sweep and a cathodic sweep, said rate of voltage variation being between 0.5 volts/min and 4 volts/min.
 11. The method of claim 1 or 2 further comprising the step of forming the ratio of said total charge to the total absolute value of charge during said portion.
 12. A method for sensing the concentration of glucose in biological fluids comprisingapplying a voltage to an electrode arranged for interaction with said fluids to cause a current to flow through said electrode, sweeping said voltage in a first direction from a first limit to a second limit and thereafter in the reverse direction, said sweep including voltages between 0.2 and 0.6 volts, measuring said current at at least one isolated point, between 0.2 volts and 0.6 volts, during the anodic variation of the voltage sweep, said point being selected to be sensitive to change in levels of glucose concentration in said fluid, but relatively insensitive to levels of other constituents of said fluid, and determining the glucose concentration level based on the current measurement at said isolated point.
 13. The method of claim 12 wherein said lower limit of said voltage variation is between -1.0 and -0.6 volts and the upper limit is between 0.7 and 1.1 volts.
 14. The method of claim 13 wherein said dwell periods are greater than 2 seconds.
 15. The method of claim 14 wherein the rate of voltage change during said cathodic sweep is more rapid than during said anodic sweep.
 16. The method of claim 12 wherein the rate of change of said voltage variation is constant between said limits, and includes dwell periods at each limit. 